Mapping the immunogenicity of therapeutic protein products

30th May 2014

Posted By Paul Boughton

The label-free Attana Cell 200
Aggregation assays. Three assays to study aggregation properties is A) off-rate screening, B) secondary antibody interaction and C) capture assay. A) The more aggregated, the slower dissociation rate due to avidity and rebinding. B) The more aggregated, more binding to a secondary antibody due to more available epitopes. C) The more aggregated, the higher frequency response due to more mass added to the surface
Off-target interactions. The frequency responses are obtained from antibody interactions to transfected (A) and not transfected (B) CHO cells. The specific antibody-receptor interaction is clearly observed in the dissociation phase (A, after 84s), while off-target interactions occurs while the antibody is continuously injected over the cells during the association phase (arrows)

Samuel Altun, Lena Heffler and Teodor Aastrup provide some examples of immunogenicity testing assays.

It is of great importance to understand and verify desired and unwanted immunogenicity effects. Immune responses to therapeutic protein products can limit product efficacy as well as affect patient safety.

Consequently, immunogenicity-related factors should be considered early in the product development process. Guidance for immunogenicity testing is provided by the U.S. Food and Drug Administration (FDA). Factors such as aggregation, off-target interactions, target clustering and neutralising antibodies are mentioned as examples that should be evaluated1.

The discussion here pertains to a proprietary quartz crystal microbalance (QCM) based biosensor that offers label-free applications to study the abovementioned factors in real time.

The Attana Cell 200 biosensor is based on the piezoelectric effect of quartz. When applying an alternating electric potential over a quartz crystal, it will start to oscillate with a resonance frequency related to the crystal's mass. Hence when any molecules are bound to the crystal, the frequency changes and the interaction can be characterised, for instance as kinetic rate constants and affinity.


Protein aggregates have been known to elicit immune responses to therapeutic protein products and are seen as an immunity-provoking factor.1 Aggregation is therefore an important factor to study during development processes and the Attana Cell 200 biosensor is an excellent tool to study it.2 Three assays of studying aggregation properties are illustrated in this chapter: A) Off-rate screening, B) Secondary binding and C) Capture assay.2

The results of the off-rate screening show the more aggregated proteins, the slower dissociation rate, due to higher avidity and rebinding.

In the secondary binding assay, more of a secondary antibody binds to the aggregated proteins, which has more epitopes available for binding and thus gives a higher shift in frequency response.

In contrast to the off-rate screening, the third assay points out the association phase, where aggregated proteins gain a higher frequency response when the surface is saturated, due to the higher loading of mass.

Receptor clustering

Clustering of receptors and antigens is a natural event and affects binding properties such as kinetic rate and affinity.3 Different protein products may interact differently to clustered receptors than non-clustered ones.1 In addition, the cell membrane with its variety of components may lead to natural interactions between the protein product and other molecules than the target antigen.

Measuring the protein binding in a cell-based assay provides an added value to the kinetic information and is more biologically relevant than measuring the protein-antigen binding in a purified biochemical assay.

In this chapter, an antibody-cell interaction study is shown to illustrate off-target interactions. In the Attana Cell 200 biosensor, CHO cells and transfected CHO cells were used to distinguish specific and off-target interactions.

The result clearly shows that the antibody interacts specifically to the receptor in the transfected cells and also to other membrane components (shown as a fast interaction giving the typical shape of a hill, arrows in Fig. 3). The data was fitted to a 1:2 binding model and the affinity calculated to 7nM (specific interaction) and 1.4µM (off-target interactions). It is important to detect and evaluate biological natural events at an early stage of the therapeutic protein product development.

This is to secure desired effects and limit unwanted immune responses against the products, both for patient safety and product efficacy.

The label-free Attana Cell 200 biosensor can be used for studies of many of the factors mentioned in the FDA guidelines.1

For more information at

Samuel Altun, Lena Heffler and Teodor Aastrup are with Attana, in Stockholm, Sweden.

Reference: 1 US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, & Center for Biologics Evaluation and Research. (2013, February). Immunogenicity Assessment for Therapeutic Protein Products. Guidance for Industry, pp. 1-32; 2 Zurdo, J, Michael, R, Rebecca, Y, Hedman, K, & Aastrup, T (2011, June). Improving the Developability. Innovations in Pharmaceutical Technology, pp. 34-40; 3 Aastrup, T (2013). Talking Sense. Innovations in Pharmaceutical Technology, pp. 48-51.





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